Polynucleotides encoding anti-sulfotyrosine antibodies

ABSTRACT

The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

RELATED APPLICATIONS

This patent application is a continuation-in-part of, and claimspriority under 35 USC 120 to, U.S. patent application Ser. No.11/328,899, filed Jan. 9, 2006, which claims priority under 35 USC 119to U.S. Provisional patent application No. 60/642,445, filed Jan. 7,2005, the entire disclosures of which are hereby incorporated byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the United States Department of Energy to TheRegents of The University of California. The government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Tyrosine sulfation is an ubiquitous post-translational modification thatoccurs in essentially all animal cells containing a Golgi apparatus(Huttner, 1998, Ann. Rev. Physiol. 50: 363-376). As much as 1% of thetyrosine residues in an organisms total protein are capable of beingsulfated. Tyrosine sulfation of proteins occurs in the trans-Golgi, andthe modification appears to be involved in intracellular transport,proteolytic processing and alteration of biological activity. Theability to detect, isolate and differentiate sulfotyrosine-containingproteins would therefore prove extremely valuable. However, no antibodycapable of discriminating tyrosine-sulfated proteins from non-sulfatedproteins has been described.

More particularly, tyrosine sulfation has been found to driveextracellular protein-protein interactions. The archetypal example isthe interaction between PSGL-1 and P-selectin, in which PSGL-1'ssulfated tyrosine residues are positioned to form hydrogen bonds withP-selectin. Additionally, the tyrosine-sulfated chemokine receptorsCCR5, CXCR4, and CCR2 rely on their sulfate groups to increase bindingaffinity for their chemokines, and in the case of CCR5 are exploited byHIV to mediate infection.

Antibodies against posttranslational modifications have proven to bevital tools for functional studies. For example, an antibody againstnitrotyrosine was recently used to identify proteins which are nitratedduring the inflammatory response, and antibodies against tyrosinephosphate have been widely used for decades. The obvious utility of anantibody against tyrosine sulfate has led a number of laboratories topursue this important tool (Bundgaard et al., 2002, Analysis ofTyrosine-O-Sulfation, Methods in Molecular Biology PosttranslationalModification of Proteins, pp. 223-239). While an antibody whose bindingepitope includes tyrosine sulfate has been reported (Snapp et al., 1998,Blood 91: 154-164), there are no reports of an antibody that recognizesonly a sulfated tyrosine residue, despite numerous unsuccessfulimmunization attempts to derive antibodies recognizing sulfatedtyrosines. One explanation for these failures is that the presence ofsulfated tyrosine residues in many secreted and membrane-bound proteinshas led vertebrate immune systems to become tolerant of themodification, rendering standard immunization-based antibody generationmethodologies useless.

One way to overcome the limitations of intact immune systems in thegeneration of specific antibodies against non-immunogenic targets is touse phage antibody libraries rather than immunization. In this techniquelarge numbers (≧10⁹) of different antibodies are displayed on thesurface of filamentous phage and specific binders are selected on thebasis of their binding abilities to target antigens. The fact that thistechnology is completely in vitro, using either natural rearranged orsynthetic V genes, overcomes the intrinsic biases of the immune system.Although phage antibodies have been selected against large numbers ofdifferent polypeptide and chemical targets, including specific peptides,there have been no descriptions of the use of this technology to selectantibodies against post-translational modifications.

There is therefore a need for antibodies capable of specificallyrecognizing tyrosine-sulfated proteins and capable of distinguishingbetween sulfated and non-sulfated proteins. The present inventionaddresses this need.

SUMMARY OF THE INVENTION

The invention provides anti-sulfotyrosine specific antibodies, antibodyfragments, and immunological methods capable of detecting and isolatingpolypeptides that are tyrosine-sulfated. Polynucleotides encoding thesulfotyrosine antibodies and antibody fragments, vectors and expressionvectors comprising such polynucleotides, and host cells used for theproduction of the antibodies and antibody fragments of the invention arealso provided. Further, methods of detecting, isolating, and quantifyingsulfotyrosine-containing proteins are provided. The sulfotyrosineantibodies and antibody fragments of the invention may be used todiscriminate between the non-sulfated and sulfated forms of suchproteins. Preferred sulfotyrosine antibodies and sulfotyrosine antibodyfragments of the invention specifically bind to a polypeptide containinga sulfated tyrosine, but do not bind or weakly bind to a polypeptidethat does not contain a sulfated tyrosine, in standard immunologicaldetection assays, including without limitation, ELISA, immunoblot,Western Blot, immunoprecipitation, and the like, under conditionstypically employed for such assays.

Using a phage-display system, single chain antibodies (scFvs) weregenerated and screened against tyrosine-sulfated synthetic peptideantigens, resulting in the isolation of scFvs that specificallyrecognize sulfotyrosine-containing peptides and/or demonstratesulfotyrosine-specific binding in tyrosine sulfated proteins (seeExamples 1 and 2, infra). The single chain antibodies provided hereinmay be multimerized or cloned into various immunoglobulin scaffolds andexpressed as full length antibodies, as is generally known. In anexemplified embodiment, a sulfotyrosine-specific IgG was generated usingthe VH and VL genes of a sulfotyrosine-specific scFv (see Examples 3 and4, infra).

The sulfotyrosine antibodies and antibody fragments of the inventionwill be useful in a wide variety of immunological protein assays andisolation procedures, including without limitation, ELISAs, WesternBlots and other immunoblot techniques, immunohistochemical assays,various affinity purification methods, and the like, and may be used inproteomics-based approaches to the identification and isolation oftyrosine-sulfated proteins, in cell-based assays of inhibitorcandidates, and in drug-screening and development endeavors, among otheruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A): structure of the tyrosine sulfate post-translationalmodification. (B): sequences of the two peptides used for selection; thetyrosine which is sulfated is indicated in bold, with the SO₃ attached.(C): Each of the peptides was coupled to ovalbumin, BSA or biotin viathe C terminal SH group. (D): amino acid sequences flanking knowntyrosine sulfate modification sites.

FIG. 2. Binding capabilities of scFvs. (A): soluble scFv ELISA resultsfor 22 of the putatively positive clones identified by phage ELISA. (B):confirmatory ELISA with the two clones scFv25 and 32 identified asbinding in a sulfate dependent manner. BSA-1S and BSA-1 representpeptide 1 coupled to BSA in the sulfated and non-sulfated forms.

FIG. 3. SDS-PAGE gel (12% Tris-HCl) showing proteins treated anduntreated with Abalone sulfatase. 15 μl of native and sulfatase treated(*) fibrinogen, IgM, and thyroglobulin per lane.

FIG. 4. ELISA results (see Example 2) using scFv 25 against sulfated andunsulfated (Abalone sulfatase digested) proteins and peptides.

FIG. 5. Polyacrylamide gel electrophoresis of the proteins used in theELISA (See FIG. 4), showing the effects of sulfatase treatment.

FIG. 6. (A): ELISAs carried out with full length IgG against a number ofsulfated proteins. (B): ELISAs carried out with the scFv-AP fusionprotein. (C): Polyacrylamide gel electrophoresis of the analyzedproteins before and after sulfatase treatment.

FIG. 7. (A): Inhibition of IgG binding to bovine fibrinogen IV uponincubation with tyrosine sulfate, but not tyrosine or tyrosinephosphate. (B): as A, except the scFv-AP fusion was used. (C): Titrationof the inhibition of IgG binding to fibrinogen IV by increasingconcentrations of tyrosine sulfate.

FIG. 8. (A): Polyacrylamide gel electrophoresis of different proteinstreated (lanes 5, 7, 9, 11 and 13), and not (lanes 1, 4, 6, 8, 10 and12), with abalone sulfatase. (B): the same proteins as in (A) analyzedby Western Blotting using the IgG (lanes 1-11) or scFv-AP fusion (12 and13). Lanes 1) E. coli extract; 2) abalone sulfatase; 3) Markers; 4 & 5)Fibrinogen 1S; 6 & 7) Fibrinogen IV; 8 & 9) Rat fibrinogen; 10 & 11)human C4; 12 & 13) vitronectin.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides antibodies, antibody fragments, and immunologicalmethods capable of detecting and isolating polypeptides that aretyrosine-sulfated. Polynucleotides encoding the sulfotyrosine antibodiesand antibody fragments, vectors and expression vectors comprising suchpolynucleotides, and host cells used for the production of theantibodies and antibody fragments of the invention are also provided.Further, methods of detecting, isolating, and quantifyingsulfotyrosine-containing proteins are provided. The sulfotyrosineantibodies and antibody fragments of the invention may be used todiscriminate between the non-sulfated and sulfated forms of suchproteins. Preferred sulfotyrosine antibodies and sulfotyrosine antibodyfragments of the invention specifically bind to a polypeptide containinga sulfated tyrosine, but do not bind or weakly bind to a polypeptidethat does not contain a sulfated tyrosine, in standard immunologicaldetection assays, including without limitation, ELISA, immunoblot,Western Blot, immunoprecipitation, and the like, under conditionstypically employed for such assays.

To date, no antibody or other tool capable of specifically identifyingproteins containing sulfated tyrosine has been described, representing afundamental obstacle in the study of this important post-translationalmodification. The present invention provides antibodies whichspecifically recognize and bind sulfotyrosine residues in both syntheticpeptides and sulfated proteins, but which do not bind to the unsulfatedcounterpart peptides or de-sulfated counterpart proteins.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and CurrentProtocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons,Inc. 2001. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and includes monoclonal antibodies, polyclonalantibodies, multivalent antibodies, and multi specific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity), regardless of how they are produced (i.e., usingimmunization, recombinant, synthetic methodologies).

The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light” chain,domain, region and component are used interchangeably, are abbreviatedby “VL” or “V_(L)” and refer to the light chain of an antibody orantibody fragment. Similarly, terms “variable heavy” chain, domain,region and component are used interchangeably, are abbreviated by “VH”or “V_(H)” and refer to the heavy chain of an antibody or antibodyfragment.

The terms “anti-sulfotyrosine antibody” and “sulfotyrosine antibody” areused interchangeably and refer to antibodies that are specific for andbind specifically to a sulfated tyrosine antigenic determinant in asulfotyrosine-containing polypeptide.

A “sulfated tyrosine antigenic determinant” may be an antigenicdeterminant located entirely within the sulfated tyrosine residue, ormay comprise the sulfated tyrosine residue and at least a part of one ormore amino acid residues within the polypeptide.

As used herein, the terms “specific”, “specifically reactive”, “specificbinding”, “specifically binds” and “binds specifically” when used inconnection with the antibodies and antibody fragments of the inventionrefer to the selective binding of sulfotyrosine antibodies andsulfotyrosine antibody fragments to a sulfated tyrosine antigenicdeterminant in a polypeptide, generally as determined using standardimmunological detection assays, including without limitation ELISA,immunoblot, Western Blot, immunohistochemical and immunoprecipitationassays, under conditions typically employed for conducting such assays,but are not bind or bind weakly to polypeptides that do not contain asulfated tyrosine. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

Antibodies and antibody fragments may be tested for such specificityusing methods well know in the art and as described herein. For example,in general, specificity may be established by comparing binding toappropriate (sulfotyrosine) antigen with binding to an irrelevantantigen or antigen mixture under a given set of conditions. As usedherein, a “sulfotyrosine antigen” is a peptide or polypeptide thatincludes a sulfated tyrosine residue. If the antibody binds to thesulfotyrosine antigen at least two times more (or, two times asstrongly) than to irrelevant antigen or antigen mixture, then it isconsidered to be specific. In one embodiment, sulfotyrosine antigen is asynthetic peptide containing at least one sulfated tyrosine residue, andthe irrelevant antigen is a synthetic peptide which is identical exceptthat the tyrosine residue(s) is (are) not sulfated. Two such pairs ofpeptides which differ only in respect of the sulfation state of thetyrosine residue contained therein (i.e., either sulfated or notsulfated) are provided herein, see Example 1, infra, and SEQ ID NOS:9-12. In another embodiment, sulfotyrosine antigen is a proteincontaining a sulfated tyrosine residue, and the irrelevant antigen isthe same protein which has been treated to remove sulfate from thetyrosine residue (i.e., using a sulfatase enzyme). Examples of suchproteins include without limitation IgM, thyroglobin, and fibrinogen.Additional proteins which may used to define specificity includesulfotyrosine containing proteins which bind specifically to scFv 25 asprovided infra (using enzymatically de-sulfated protein as theirrelevant antigen). Further, irrelevant antigens may comprisepolypeptides containing a phosphated and/or nitosylated tyrosineresidue(s) but not a sulfated tyrosine residue. Preferred sulfotyrosineantibodies of the invention are those which demonstrate specificity toat least two different sulfotyrosine antigens.

The terms “sulfotyrosine”, “tyrosine sulfate” and “sulfated tyrosine”are used interchangeably and refer to O-sulfate modified tyrosine.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgA-1, IgA-2; and etc. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably, to refer to an antibody in itssubstantially intact form, not as antibody fragments as defined below.The terms particularly refer to an antibody with heavy chains thatcontain the Fc region. A full length antibody can be a native sequenceantibody or an antibody variant.

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CH1 domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment which consists of a VH domain; (vii) isolated CDRregions; (viii) F(ab′)₂ fragments, a bivalent fragment including twoFab′ fragments linked by a disulfide bridge at the hinge region; (ix)single chain antibody molecules (e.g. single chain Fv; scFv); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain; (xi) “linear antibodies” comprising a pairof tandem Fd, segments (VH-CH1-VH-CH1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain which may be generated by papain digestion ofan intact antibody. The Fc region may be a native sequence Fc region ora variant Fc region. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue at aboutposition Cys226, or from about position Pro230, to the carboxyl-terminusof the Fc region. The Fc region of an immunoglobulin generally comprisestwo constant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain.

As used herein, the term “single-chain Fv” or “scFv” or “single chain”antibody refers to antibody fragments comprising the VH and VL domainsof antibody, wherein these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the VH and VL domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The terms “anti-sulfotyrosine antibody fragment” and “sulfotyrosineantibody fragment” are used interchangeably and refer to antibodyfragments that specifically bind to a sulfated tyrosine antigenicdeterminant in a polypeptide. The terms “anti-sulfotyrosine scFv”,anti-sulfotyrosine single chain antibody”, “sulfotyrosine scFv”, and“sulfotyrosine single chain antibody” are used interchangeably and referto single chain antibodies that specifically bind to a sulfated tyrosineantigenic determinant in a polypeptide.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. The monoclonal antibodies of the invention maybe generated by recombinant DNA methods, and are sometimes referred toas “recombinant antibodies” or “recombinant monoclonal antibodies”herein.

Recombinant antibody fragments may be isolated from phage antibodylibraries using techniques well known in the art. See, for example,Clackson et al., 1991, Nature 352: 624-628; Marks et al., 1991, J. Mol.Biol. 222: 581-597. Recombinant antibody fragments may be derived fromlarge phage antibody libraries generated by recombination in bacteria(Sblattero and Bradbury, 2000, Nature Biotechnology 18:75-80; and asdescribed herein). Polynucleotides encoding the VH and VL components ofantibody fragments (i.e., scFv) may be used to generate recombinant fulllength immunoglobulins using methods known in the art (see, for example,Persic et al., 1997, Gene 187: 9-18).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

As used herein, the term “bispecific antibody” refers to an antibody,typically a monoclonal antibody, having binding specificities for atleast two different antigenic epitopes. In one embodiment, the epitopesare from the same antigen. In another embodiment, the epitopes are fromtwo different antigens. Methods for making bispecific antibodies areknown in the art. For example, bispecific antibodies can be producedrecombinantly using the co-expression of two immunoglobulin heavychain/light chain pairs. See, e.g., Milstein et al., Nature 305:537-39(1983). Alternatively, bispecific antibodies can be prepared usingchemical linkage. See, e.g., Brennan, et al., Science 229:81 (1985).Bispecific antibodies include bispecific antibody fragments. See, e.g.,Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993),Gruber, et al., J. Immunol. 152:5368 (1994).

An “affinity matured” antibody is one with one or more modifications(mutations) in one or more CDRs thereof which result in an improvementin the affinity of the antibody for antigen, compared to the unmodifiedparent antibody. Preferred affinity matured antibodies will havenanomolar or even picomolar affinities for the target antigen. Affinitymatured antibodies are produced by various procedures known in the art,including by variable domain shuffling (see, e.g., Marks et al. 1992,Bio/Technology 10:779-783), random mutagenesis of CDR and/or frameworkresidues (see, e.g., Barbas et al., 1994, Proc Nat. Acad. Sci, USA91:3809-3813; Schier et al., 1995, Gene 169:147-155; Yelton et al.,1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol.154(7):3310-9; and, Hawkins et al, 1992, J. Mol. Biol. 226:889-896).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof (“polynucleotides”) in eithersingle- or double-stranded form. Unless specifically limited, the term“polynucleotide” encompasses nucleic acids containing known analogues ofnatural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g. degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19: 5081;Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al.,1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The termnucleic acid is used interchangeably with gene, cDNA, and mRNA encodedby a gene.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Forexample, one type of vector is a plasmid, a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a phage vector. Another type of vector is a viral vector,wherein additional DNA segments may be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or “expression vectors”.

The term “host cell” (or “recombinant host cell”), as used herein,refers to a cell that has been genetically altered, or is capable ofbeing genetically altered by introduction of an exogenouspolynucleotide, such as a recombinant plasmid or vector, and includesnot only the particular subject cell but also the progeny thereof.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term “host cell” as used herein.

The term “link” as used herein refers to a physical linkage as well aslinkage that occurs by virtue of co-existence within a biologicalparticle, e.g., phage, bacteria, yeast or other eukaryotic cell.

“Physical linkage” refers to any method known in the art forfunctionally connecting two molecules (which are termed “physicallylinked”), including without limitation, recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, covalent bonding (e.g., disulfide bonding and othercovalent bonding), hydrogen bonding; electrostatic bonding; andconformational bonding, e.g., antibody-antigen, and biotin-avidinassociations.

“Fused” refers to linkage by covalent bonding.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as VH and VL genes orpolypeptides (i.e., in a scFv), and serves to place the two molecules ina preferred configuration.

The term “isolated” refers to material which is substantially oressentially free from components which normally accompany the materialas it is found in its native or natural state. However, the term“isolated” is not intended refer to the components present in anelectrophoretic gel or other separation medium. An isolated component isfree from such separation media and in a form ready for use in anotherapplication or already in use in the new application/milieu. An“isolated” antibody is one that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The terms “label” and “detectable label” refer to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” or “detectably labeled” antibody.The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. A great number of such labels are known in the art,including without limitation protein tags, radioisotopes, metalchelators, enzymes, fluorescent compounds (dyes, proteins, chemicals),bioluminescent compounds, and chemiluminescent compounds.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, a nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a nucleic acid encoding afluorescent protein from one source and a nucleic acid encoding apeptide sequence from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testsequence. Preferably, the identity exists over a region that is at leastabout 22 amino acids or nucleotides in length, or more preferably over aregion that is 30, 40, or 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homologyalignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443,by the search for similarity method of Pearson & Lipman, 1988, Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc.Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 are used, typically withthe default parameters described herein, to determine percent sequenceidentity for the nucleic acids and proteins of the invention. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, 1993,Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “as determined by maximal correspondence” in the context ofreferring to a reference SEQ ID NO means that a sequence is maximallyaligned with the reference SEQ ID NO over the length of the referencesequence using an algorithm such as BLAST set to the default parameters.Such a determination is easily made by one of skill in the art.

A “display vector” refers to a vector used to create a cell or virusthat displays, i.e., expresses a display protein comprising aheterologous polypeptide, on its surface or in a cell compartment suchthat the polypeptide is accessible to test binding to target moleculesof interest, such as antigens.

A “display library” refers to a population of display vehicles, often,but not always, cells or viruses. The “display vehicle” provides boththe nucleic acid encoding a peptide as well as the peptide, such thatthe peptide is available for binding to a target molecule and further,provides a link between the peptide and the nucleic acid sequence thatencodes the peptide. Various “display libraries” are known to those ofskill in the art and include libraries such as phage, phagemids, yeastand other eukaryotic cells, bacterial display libraries, plasmid displaylibraries as well as in vitro libraries that do not require cells, forexample ribosome display libraries or mRNA display libraries, where aphysical linkage occurs between the mRNA or cDNA nucleic acid, and theprotein encoded by the mRNA or cDNA.

A “phage expression vector” or “phagemid” refers to any phage-basedrecombinant expression system for the purpose of expressing a nucleicacid sequence in vitro or in vivo, constitutively or inducibly, in anycell, including prokaryotic, yeast, fungal, plant, insect or mammaliancell. A phage expression vector typically can both reproduce in abacterial cell and, under proper conditions, produce phage particles.The term includes linear or circular expression systems and encompassesboth phage-based expression vectors that remain episomal or integrateinto the host cell genome.

A “phage display library” refers to a “library” of bacteriophages onwhose surface is expressed exogenous peptides or proteins. The foreignpeptides or polypeptides are displayed on the phage capsid outersurface. The foreign peptide can be displayed as recombinant fusionproteins incorporated as part of a phage coat protein, as recombinantfusion proteins that are not normally phage coat proteins, but which areable to become incorporated into the capsid outer surface, or asproteins or peptides that become linked, covalently or not, to suchproteins. This is accomplished by inserting an exogenous nucleic acidsequence into a nucleic acid that can be packaged into phage particles.Such exogenous nucleic acid sequences may be inserted, for example, intothe coding sequence of a phage coat protein gene. If the foreignsequence is “in phase” the protein it encodes will be expressed as partof the coat protein. Thus, libraries of nucleic acid sequences, such asa genomic library from a specific cell or chromosome, can be so insertedinto phages to create “phage libraries.” As peptides and proteinsrepresentative of those encoded for by the nucleic acid library aredisplayed by the phage, a “peptide-display library” is generated. Whilea variety of bacteriophages are used in such library constructions,typically, filamentous phage are used (Dunn, 1996 Curr. Opin.Biotechnol. 7:547-553). See, e.g., description of phage displaylibraries, below.

The term “amplification” means that the number of copies of apolynucleotide is increased.

Preferred Embodiments

In one aspect, the invention relates to sulfotyrosine antibodies. Thesulfotyrosine antibodies of the invention are specific for a sulfatedtyrosine antigenic determinant in a sulfotyrosine-containingpolypeptide. Sulfotyrosine antibodies may be polyclonal, monoclonal, andmay be produced by recombinant means or in cells derived fromimmunizations. Preferred sulfotyrosine antibodies are isolated, purifiedor semi-purified such that they retain specificity in the desiredapplication. In one embodiment, a sulfotyrosine antibody of theinvention comprises a heavy chain variable region having an amino acidsequence that is at least 80%, preferably about 90%, 91%, 92%, 93% or94%, and most preferably about 95% or more, identical to the amino acidsequence of SEQ ID NO: 8. In another embodiment, a sulfotyrosineantibody of the invention comprises a light chain variable region havingan amino acid sequence that is at least 80%, preferably about 90%, 91%,92%, 93% or 94%, and most preferably about 95% or more, identical to theamino acid sequence of SEQ ID NO: 4. Yet another embodiment is asulfotyrosine antibody comprising a heavy chain variable region havingan amino acid sequence that is at least 80%, preferably about 90%, 91%,92%, 93% or 94%, and most preferably about 95% or more, identical to theamino acid sequence of SEQ ID NO: 8, and a light chain variable regionan amino acid sequence that is at least 80%, preferably about 90%, 91%,92%, 93% or 94%, and most preferably about 95% or more, identical to theamino acid sequence of SEQ ID NO: 4. In another embodiment, asulfotyrosine antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO: 8, and a light chain variableregion having the amino acid sequence of SEQ ID NO: 4. The sulfotyrosineantibodies of the invention may be of the immunoglobulin classes IgA,IgD, IgE, IgG and IgM and subclasses thereof.

A related aspect of the invention relates to sulfotyrosine antibodyfragments. The sulfotyrosine antibody fragments of the invention arespecific for a sulfated tyrosine antigenic determinant in asulfotyrosine-containing polypeptide. Such fragments may be generatedfrom intact antibodies or through the use of recombinant technology. Forexample, in one embodiment, a recombinant sulfotyrosine antibodyfragment is a single chain antibody or scFv. In a related embodiment, arecombinant sulfotyrosine antibody fragment is a single chain an Fab orFab′ fragment. Such recombinant sulfotyrosine antibody fragments maycomprise a heavy chain variable region having an amino acid sequencethat is at least 80%, preferably about 90%, 91%, 92%, 93% or 94%, andmost preferably about 95% or more, identical to the amino acid sequenceof SEQ ID NO: 8. In another embodiment, a recombinant sulfotyrosineantibody fragment comprises a light chain variable region having anamino acid sequence that is at least 80%, preferably about 90%, 91%,92%, 93% or 94%, and most preferably about 95% or more, identical to theamino acid sequence of SEQ ID NO: 4. Yet another embodiment is arecombinant sulfotyrosine antibody fragment comprising a heavy chainvariable region having an amino acid sequence that is at least 80%,preferably about 90%, 91%, 92%, 93% or 94%, and most preferably about95% or more, identical to the amino acid sequence of SEQ ID NO: 8, and alight chain variable region an amino acid sequence that is at least 80%,preferably about 90%, 91%, 92%, 93% or 94%, and most preferably about95% or more, identical to the amino acid sequence of SEQ ID NO: 4.

The sulfotyrosine antibodies and antibody fragments of the invention maybe detectably labeled as is generally known. The label may be detectableby itself (e.g. radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable. A great number ofsuch labels are known in the art, including without limitation proteintags, radioisotopes, metal chelators, enzymes, fluorescent compounds(dyes, proteins, chemicals), bioluminescent compounds, andchemiluminescent compounds.

Sulfotyrosine-specific scFvs may be isolated using selection andscreening strategies which utilize sulfotyrosine antigens. A number ofselection and screening strategies may be adopted for isolating singlechain sulfotyrosine antibody fragments. Diversity libraries may, forexample, be generated using phage display or other display methods. Inone embodiment, one or more pairs of such sulfotyrosine antigens areemployed, in combination with paired “irrelevant” antigens. Examples ofsuch sulfotyrosine and irrelevant antigens include but are not limitedto the peptides of SEQ ID NOS: 9-12. Exemplary selection and screeningstrategies are described in the Examples, infra.

As further described in Example 1, infra, libraries of phage-displayedsingle chain variable fragments (scFvs) containing natural combinationsof heavy and light chain variable regions were used to select scFvsrecognizing tyrosine sulfate. As illustrated in the Examples, severalscFvs which bind specifically or preferentially to synthetic peptidescontaining a sulfated tyrosine were isolated and studied. For example,clone scFv 25 appears to be highly specific for the sulfotyrosinemodification in both the synthetic peptides and in tyrosine-sulfatedproteins.

Construction of phage display libraries exploits the bacteriophage'sability to display peptides and proteins on their surfaces, i.e., ontheir capsids. Often, filamentous phage such as M13, fd, or f1 are used.Filamentous phage contain single-stranded DNA surrounded by multiplecopies of genes encoding major and minor coat proteins, e.g., pIII. Coatproteins are displayed on the capsid's outer surface. DNA sequencesinserted in-frame with capsid protein genes are co-transcribed togenerate fusion proteins or protein fragments displayed on the phagesurface. Phage libraries thus can display peptides representative of thediversity of the inserted sequences. Significantly, these peptides canbe displayed in “natural” folded conformations. The fluorescent bindingligands expressed on phage display libraries can then bind targetmolecules, i.e., they can specifically interact with binding partnermolecules such as antigens, e.g., (Petersen, 1995, Mol. Gen. Genet.,249:425-31), cell surface receptors (Kay, 1993, Gene 128:59-65), andextracellular and intracellular proteins (Gram, 1993, J. Immunol.Methods, 161:169-76).

The concept of using filamentous phages, such as M13 or fd, fordisplaying peptides on phage capsid surfaces was first introduced bySmith, 1985, Science 228:1315-1317. Peptides have been displayed onphage surfaces to identify many potential ligands (see, e.g., Cwirla,1990, Proc. Natl. Acad. Sci. USA, 87:6378-6382). There are numeroussystems and methods for generating phage display libraries described inthe scientific and patent literature, see, e.g., Sambrook and Russell,Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press, Chapter 18, 2001; Phage, Display of Peptides andProteins: A Laboratory Manual, Academic Press, San Diego, 1996; Crameri,1994, Eur. J. Biochem. 226:53-58; de Kruif, 1995, Proc. Natl. Acad. Sci.USA, 92:3938-42; McGregor, 1996, Mol. Biotechnol., 6:155-162; Jacobsson,1996, Biotechniques, 20:1070-1076; Jespers, 1996, Gene, 173:179-181;Jacobsson, 1997, Microbiol Res., 152:121-128; Fack, 1997, J. Immunol.Methods, 206:43-52; Rossenu, 1997, J. Protein Chem., 16:499-503; Katz,1997, Annu. Rev. Biophys. Biomol. Struct., 26:27-45; Rader, 1997, Curr.Opin. Biotechnol., 8:503-508, Griffiths, 1998, Curr. Opin. Biotechnol.,9:102-108.

Typically, exogenous nucleic acids encoding the protein sequences to bedisplayed are inserted into a coat protein gene, e.g. gene III or geneVIII of the phage. The resultant fusion proteins are displayed on thesurface of the capsid. Protein VIII is present in approximately 2700copies per phage, compared to 3 to 5 copies for protein III (Jacobsson(1996), supra). Multivalent expression vectors, such as phagemids, canbe used for manipulation of the nucleic acid sequences encoding thefluorescent binding library and production of phage particles inbacteria (see, e.g., Felici, 1991, J. Mol. Biol., 222:301-310).

Phagemid vectors are often employed for constructing the phage library.These vectors include the origin of DNA replication from the genome of asingle-stranded filamentous bacteriophage, e.g., M13 or f1 and requirethe supply of the other phage proteins to create a phage. This isusually supplied by a helper phage which is less efficient at beingpackaged into phage particles. A phagemid can be used in the same way asan orthodox plasmid vector, but can also be used to produce filamentousbacteriophage particle that contain single-stranded copies of clonedsegments of DNA.

The displayed protein does not need to be a fusion protein. For example,a fluorescent binding ligand may attach to a coat protein by virtue of anon-covalent interaction, e.g., a coiled coil binding interaction, suchas jun/fos binding, or a covalent interaction mediated by cysteines(see, e.g., Crameri et al., 1994, Eur. J. Biochem., 226:53-58) with orwithout additional non-covalent interactions. Morphosys have described adisplay system in which one cysteine is put at the C terminus of thescFv or Fab, and another is put at the N terminus of g3p. The twoassemble in the periplasm and display occurs without a fusion gene orprotein.

The coat protein does not need to be endogenous. For example, DNAbinding proteins can be incorporated into the phage/phagemid genome(see, e.g., McGregor & Robins, 2001, Anal. Biochem., 294:108-117,). Whenthe sequence recognized by such proteins is also present in the genome,the DNA binding protein becomes incorporated into the phage/phagemid.This can serve as a display vector protein. In some cases it has beenshown that incorporation of DNA binding proteins into the phage coat canoccur independently of the presence of the recognized DNA signal.

Other phage can also be used. For example, T7 vectors, T4 vector, T2vectors, or lambda vectors can be employed in which the displayedproduct on the mature phage particle is released by cell lysis.

Another methodology is selectively infective phage (SIP) technology.which provides for the in vivo selection of interacting protein-ligandpairs. A “selectively infective phage” consists of two independentcomponents. For example, a recombinant filamentous phage particle ismade non-infective by replacing its N-terminal domains of gene 3 protein(g3p) with a protein of interest, e.g., an antigen. The nucleic acidencoding the antigen can be inserted such that it will be expressed. Thesecond component is an “adapter” molecule in which the fluorescentligand is linked to those N-terminal domains of g3p that are missingfrom the phage particle. Infectivity is restored when the displayedprotein (e.g., a fluorescent binding ligand) binds to the antigen. Thisinteraction attaches the missing N-terminal domains of g3p to the phagedisplay particle. Phage propagation becomes strictly dependent on theprotein-ligand interaction. See, e.g., Spada, 1997, J. Biol. Chem.378:445-456; Pedrazzi, 1997, FEBS Lett. 415:289-293; Hennecke, 1998,Protein Eng. 11:405-410.

In addition to phage display libraries, analogous epitope displaylibraries can also be used. For example, the methods of the inventioncan also use yeast surface displayed libraries (see, e.g., Boder, 1997,Nat. Biotechnol., 15:553-557 and Feldhaus et al., 2003, Nat.Biotechnol., 21, 163-170), which can be constructed using such vectorsas the pYD1 yeast expression vector. Other potential display systemsinclude mammalian display vectors and E. coli libraries.

In vitro display library formats known to those of skill in the art canalso be used, e.g., ribosome displays libraries and mRNA displaylibraries. In these in vitro selection technologies, proteins are madeusing cell-free translation and physically linked to their encoding mRNAafter in vitro translation. In typical methodology for generating theselibraries, DNA encoding the sequences to be selected are transcribed invitro and translated in a cell-free system.

In ribosome display libraries (see, e.g., Mattheakis et al., 1994, Proc.Natl. Acad. Sci. USA 91:9022-9026; Hanes & Pluckthrun, 1997, Proc. Natl.Acad. Sci. USA, 94:4937-4942) the link between the mRNA encoding theantibody fragment of the invention and the ligand is the ribosomeitself. The DNA construct is designed so that no stop codon is includedin the transcribed mRNA. Thus, the translating ribosome stalls at theend of the mRNA and the encoded protein is not released. The encodedprotein can fold into its correct structure while attached to theribosome. The complex of mRNA, ribosome and protein is then directlyused for selection against an immobilized target. The mRNA from boundribosomal complexes is recovered by dissociation of the complexes withEDTA and amplified by RT-PCR.

Method and libraries based on mRNA display technology, also referred toherein as puromycin display, are described, for example in U.S. Pat.Nos. 6,261,804; 6,281,223; 6,207,446; and 6,214553. In this technology,a DNA linker attached to puromycin is first fused to the 3′ end of mRNA.The protein is then translated in vitro and the ribosome stalls at theRNA-DNA junction. The puromycin, which mimics aminoacyl tRNA, enters theribosomal A site and accepts the nascent polypeptide. The translatedprotein is thus covalently linked to its encoding mRNA. The fusedmolecules can then be purified and screened for binding activity. Thenucleic acid sequences encoding ligands with binding activity can thenbe obtained, for example, using RT-PCR.

Plasmid display systems rely on the fusion of displayed proteins to DNAbinding proteins, such as the lac repressor (see, e.g., Gates et al.,1996, J. Mol. Biol., 255:373-386; 1996, Methods Enzymol. 267:171-191).When the lac operator is present in the plasmid as well, the DNA bindingprotein binds to it and can be co-purified with the plasmid. Librariescan be created linked to the DNA binding protein, and screened uponlysis of the bacteria. The desired plasmid/proteins are rescued bytransfection, or amplification.

Methods of screening diversity libraries are well known to those in theart. The libraries are typically screened using an antigen, or moleculeof interest, for which it is desirable to select a binding partner.Typically, the antigen is attached to a solid surface or a specific tag,such as biotin. The antigen (or molecule of interest) is incubated witha library of the invention. Those polypeptides that bind to the antigenare then separated from those that do not using any of a number ofdifferent methods. These methods involve washing steps, followed byelution steps. Washing can be done, for example, with PBS, ordetergent-containing buffers. Elution can be performed with a number ofagents, depending on the type of library. For example, an acid, a base,bacteria, or a protease can be used when the library is a phage displaylibrary.

To facilitate the identification and isolation of the antigen-boundrecombinant single chain antibodies of the invention, the single chainantibody can also be engineered as a fusion protein to include selectionmarkers (e.g., epitope tags). Antibodies reactive with the selectiontags present in the fusion proteins or moieties that bind to the labelscan then be used to isolate the antigen-single chain antibody complexvia the epitope or label. For example, scFv/antigen complexes can beseparated from non-complexed display particles using antibodies specificfor the antibody selection “tag” e.g., an SV5 antibody specific to anSV5 tag (see Example 1, infra). In libraries that are constructed usinga display vector, such as a phage display vector, the selected clones,e.g., phage, are then used to infect bacteria.

Other detection and purification facilitating domains include, e.g.,metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, or the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp, Seattle Wash.). Any epitope with acorresponding high affinity antibody can be used, e.g., a myc tag (see,e.g., Kieke, 1997, Protein Eng. 10:1303-1310), V5 (Invitrogen), or anE-tag (Pharmacia). See also Maier, 1998, Anal. Biochem. 259:68-73;Muller, 1998, Anal. Biochem. 259:54-61. The inclusion of a cleavablelinker sequences such as Factor Xa, tobacco etch virus protease orenterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and binding site may be useful to facilitate purification. Forexample, an expression vector of the invention may include apolypeptide-encoding nucleic acid sequence linked to six histidineresidues. A widely used tags is six consecutive histidine residues or6His tag. These residues bind with high affinity to metal ionsimmobilized on chelating resins even in the presence of denaturingagents and can be mildly eluted with imidazole. Selection tags can alsomake the epitope or binding partner (e.g., antibody) detectable oreasily isolated by incorporation of, e.g., predetermined polypeptideepitopes recognized by a secondary reporter/binding molecule, e.g.,leucine zipper pair sequences; binding sites for secondary antibodies;transcriptional activator polypeptides; and other selection tag bindingcompositions. See also, e.g., Williams, 1995, Biochemistry,34:1787-1797.

Once a recombinant sulfotyrosine antibody fragment, such as an scFv, isselected, the nucleic acid encoding it is readily obtained. Thissequence may then be expressed using any of a number of systems toobtain the desired quantities of the protein. There are many expressionsystems for that are well know to those of ordinary skill in the art.(See, e.g., Gene Expression Systems, Fernandes and Hoeffler, Eds.Academic Press, 1999; Ausubel, supra). Typically, the polynucleotideencoding the sulfotyrosine antibody or antibody fragment is placed underthe control of a promoter that is functional in the desired host cell.An extremely wide variety of promoters are available, and can be used inthe expression vectors of the invention, depending on the particularapplication. Ordinarily, the promoter selected depends upon the cell inwhich the promoter is to be active. Other expression control sequencessuch as ribosome binding sites, transcription termination sites and thelike are also optionally included.

Another aspect of the invention relates to polynucleotides encoding thesulfotyrosine antibodies and antibody fragments of the invention, aswell as vectors and expression vectors comprising such polynucleotides.The polynucleotides and expression vectors of the invention are usefulfor the production of the antibodies and antibody fragment of theinvention.

Recombinant methods of producing the sulfotyrosine antibodies andsulfotyrosine antibody fragments of the invention are preferred.Sulfotyrosine antibody and antibody fragment encoding polynucleotidesmay be inserted into vectors capable of directing the expression of thedesired antibody product in both prokaryotic and eukaryotic host cells.A number of antibody expression vectors have been described, and methodsfor generating antibodies and antibody fragments are well known in theart. See, for example, Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES(Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (OxfordUniversity Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (JohnWiley & Sons, most recent edition).

In one embodiment, phage display systems are used to select single chainantibodies specific for sulfotyrosine. Once isolated, polynucleotidesencoding specific sulfotyrosine scFvs may be cloned into expressionvectors designed to express full length immunoglobulins as well asfragments thereof having the same specificity. Briefly, the V_(H) andV_(L) genes of the single chain antibody are cloned into animmunoglobulin scaffold (i.e., IgG) vector, expressed, and dimerized inorder to ‘convert’ the single chain into a full antibody. Theimmunoglobulin scaffold may be selected from any of the five majorclasses of immunoglobulins (IgA, IgD, IgE, IgG and IgM), and subclassesthereof (i.e., IgG-1). Example 3, infra, describes the generation of afull length, anti-sulfotyrosine IgG using the VH and VL genes of ananti-sulfotyrosine scFv. This antibody, like the scFv, also demonstratessulfotyrosine specificity (see Example 4, infra). Recombinantsulfotyrosine antibodies which are to be used to detect or isolatetyrosine sulfated proteins may be generated from immunoglobulinscaffolds of any vertebrate origin. Recombinant sulfotyrosine antibodieswhich are to be used therapeutically or diagnostically in an animal invivo should be based on an immunoglobulin scaffold that matches theimmunoglobulins of the animal.

Methods for the conversion of scFvs into intact immunoglobulin moleculesare well known, and include without limitation, the methods andexpression vectors described in Persic et al., 1997, An integratedvector system for the eukaryotic expression of antibodies or theirfragments after selection from phage display libraries. Gene 187: 9-18).See also, WO 94/11523, WO 97/9351, EP 0481790.

A sulfotyrosine antibody of the invention may be modified to increasebinding affinity, improve stability, and the like, using standardtechniques. For example, substitutions, deletions and insertions ofamino acids in the antibody polypeptides may be introduced (see, infra).

Based on the specificity for tyrosine sulfate exhibited by two scFvs andthe full length IgG described in the Examples, infra, it is notnecessary that the recombinant antibodies of the invention beglycosylated or expressed in eukaryotic cells. Various prokaryoticexpression host cells may therefore be useful in the generation of suchrecombinant sulfotyrosine antibodies and fragments. Bacterial expressionsystems are preferred, and a wide variety of appropriate expressionvectors and methods are know. E. coli host cells are preferred.

In bacterial expression systems, the expressed light and heavy chainpolypeptides of the present invention are secreted into and recoveredfrom the periplasm of the host cells. Protein recovery typicallyinvolves disrupting the microorganism, generally by such means asosmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

In one aspect of the invention, the antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD.sub.550 of about 180-220, at which stagethe cells are in the early stationary phase. A variety of inducers maybe used, according to the vector construct employed, as is known in theart and described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et at.(1999) J. Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210. Sufficient disulfide bonds are particularlyimportant for the formation and folding of full length, bivalentantibodies having two heavy chains and two light chains.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease 1, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In a preferred embodiment, E. coli strains deficient for proteolyticenzymes and transformed with plasmids overexpressing one or morechaperone proteins are used as host cells in the expression system ofthe invention. Some of these strains are further described in theExamples section below.

Heavy and light chains may be expressed from a single construct or usingmultiple constructs (expression vectors). For example, United Statespatent application of Simmons et al., No. 20050170464, describes aprocess for producing an immunoglobulin in a prokaryotic host cell,using a “separate cistron” expression vector containing a firstpromoter-cistron pair for expression of an immunoglobulin light chainand a second promoter-cistron pair for expression of an immunoglobulinheavy chain, whereby expression of the light chain and heavy chain areindependently regulated by separate promoters. Each cistron within theexpression cassette polynucleotide comprises a translation initiationregion (TIR) operably linked to the nucleic acid sequence coding for thelight chain or heavy chain of the full length antibody. According tothis method, the TIR sequences within the expression vector of theinvention are manipulated so to provide different translational strengthcombinations for light and heavy chains.

When using recombinant techniques, the antibody can be producedintracellularly or in the periplasmic space, or directly secreted intothe medium. If the antibody is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology, 10: 163-167 (1992) describes a procedure forisolating antibodies that are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an AMICON™ or MILLIPORE PELLICON™ultrafiltration unit. A protease inhibitor such as phenylmethylsulphonylfluoride (PMSF) may be included in any of the foregoing steps to inhibitproteolysis, and antibiotics may be included to prevent the growth ofadventitious contaminants.

In another aspect of the invention, the variable heavy and light chainsof recombinant sulfotyrosine antibody fragments may be multimerized(i.e., scFvs may be multimerized) to increase binding affinity, by forexample, in vitro biotinylation and avidin capture to isolate tetramers.Various strategies have been developed for preparing scFv as amultimeric derivative. This is intended to lead, in particular, torecombinant antibodies with increased binding avidity. In order toachieve multimerization of the scFv, scFv are prepared as fusionproteins with multimerization domains. The multimerization domains maybe, e.g. the CH3 region of an IgG or coiled coil structure (helixstructures) such as Leucine-zipper domains. However, there are alsostrategies in which the interaction between the VH/VL regions of thescFv are used for the multimerization (e.g. di-, tri- and pentabodies).Diabodies are a bivalent homodimeric scFv derivative (Hu et al., 1996,PNAS16: 5879-5883). The shortening of the linker in an scFv molecule to5-10 amino acids leads to the formation of homodimers in which aninter-chain VH/NL-superimposition takes place. Diabodies mayadditionally be stabilized by the incorporation of disulphide bridges.Examples of diabody-antibody proteins may be found in Perisic et al.,1994, Structure 2: 1217-1226.

By minibody the skilled person means a bivalent, homodimeric scFvderivative. It consists of a fusion protein which contains the CH3region of an immunoglobulin, preferably IgG, most preferably IgG1 as thedimerization region which is connected to the scFv via a Hinge region(e.g. also from IgG1) and a Linker region. The disulphide bridges in theHinge region are mostly formed in higher cells and not in prokaryotes.Examples of minibody-antibody proteins may be found in Hu et al., 1996,Cancer Res. 56: 3055-61. A triabody is a trivalent homotrimeric scFvderivative wherein VH-VL are fused directly without a linker sequenceleading to the formation of trimers (see, for example, Kortt et al. 1997Protein Engineering 10: 423-433).

Suitable host cells for cloning or expressing the polynucleotides in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g. B.licheniformis 41 P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC27,325) are suitable. These examples are illustrative rather thanlimiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forsulfotyrosine antibody-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated sulfotyrosineantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g. the L-1 variant ofAutographa californica NPV and the Bm-5 strain of Bombyx mori NPV, andsuch viruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc.Natl. Acad. Sci. USA, 77:4216 (1980), including DG44 (Urlaub et al.,Som. Cell and Mol. Gen., 12: 555-566 (1986)) and DP12 cell lines); mousesertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors (or these vectors are otherwiseintroduced, for example by chemical transfection methods) forsulfotyrosine antibody production and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the sulfotyrosine antibodies of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described, for example, in Ham et al., Meth. Enz. 58:44(1979); Barnes et al., Anal. Biochem., 102:255 (1980); U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 1990/03430;WO 1987/00195; or U.S. Pat. Re. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

In a preferred embodiment, the antibody protein produced herein isfurther purified to obtain preparations that are substantiallyhomogeneous for further assays and uses. Standard protein purificationmethods known in the art can be employed. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC, chromatography on silica or on a cation-exchange resin suchas DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, andgel filtration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column. Insome applications, the column has been coated with a reagent, such asglycerol, in an attempt to prevent nonspecific adherence ofcontaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the full length antibody toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the full lengthantibody is recovered from the solid phase by elution.

Another aspect of the invention relates to antibody variants. Amino acidsequence modification(s) of the sulfotyrosine antibodies and fragmentsof the invention are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the antibody are prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid alterations may be introduced in thesubject antibody amino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed full lengthantibodies are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, butframework region alterations are also contemplated.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the full length antibody of the invention, therebygenerating a Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

Antibody derivatives are also included in the invention. In this regard,the antibodies and antibody variants of the present invention can befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. Preferably, the moietiessuitable for derivatization of the antibody are water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymers areattached, they can be the same or different molecules. In general, thenumber and or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions.

Although standard immunization methods have failed to identify anantibody specific for sulfotyrosine, most probably because host animalimmune systems are tolerant of the sulfotyrosine modification,immunization methodologies may nevertheless be useful in isolatingsulfotyrosine antibodies. In this regard, appropriate antigens, i.e.,sulfotyrosine-containing peptide or protein, may be combined withvarious adjuvants which may result in the immunized host immune systemmounting an antibody response.

It is known that the secretory compartment of the cell contains proteinsthat are tyrosine sulfated. Thus, it is possible that these tyrosinesulfated proteins capture any sulfotyrosine antibodies expressed by theimmune system as they pass through the ER. One potential way ofovercoming this limitation is by amplifying the variable genes withantibody-producing cells of the immunized host, cloning the amplifiedvariable genes into antibody expression vectors and expressing theantibodies (or fragments thereby encoded), and testing for sulfotyrosinespecific reactivity as described, infra.

Specific reactivity of anti-sulfotyrosine antibodies may be establishedby a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,tyrosine sulfated proteins, peptides, tyrosine sulfatedprotein-expressing cells or extracts thereof.

In one embodiment, a pair of tyrosine-containing peptides which areidentical except in respect of the sulfation state of the tyrosine(s)therein (i.e., sulfated or not sulfated) may be employed to determinespecific reactivity. The invention provides two such peptide pairs, eachof which were chemically synthesized and contain a single tyrosineresidue (see FIG. 1). Other such peptide pairs may be generated and usedfor this purpose. The ELISA is the preferred assay format, althoughother routine immunological assays may also be employed. Althoughspecific reactivity may be determined using a single pair of suchpeptides, the use of multiple pairs of such peptides, whereintyrosine-flanking residues differ between the pairs, is preferred. In anadditional embodiment, multiple pairs of peptides are generated, such.that a number of different amino acid residues immediately flank thetyrosine residue. For example, a fully degenerate set of peptide pairsmay include all combinations of tyrosine-flanking residues in thepolymer X_(n)-Z₁-Y-Z₂-X_(n), where Z₁+Z₂ represent all combinations ofamino acids, Y is tyrosine, and X_(n) is any amino acid or polymer ofamino acids.

In another embodiment, specific reactivity may be established by using asulfotyrosine-containing protein. In this embodiment, thesulfotyrosine-containing protein is used as the relevant antigen.Irrelevant antigen is generated by de-sulfating the tyrosine(s) in thesulfotyrosine-containing protein, which may be accomplished by the useof a sulfatase enzyme. Specificity is established where the antibody orantibody fragment being assayed binds at least two times more stronglyto the relevant antigen in comparison to the irrelevant antigen.

These sulfotyrosine specific antibodies of the invention are useful forthe detection, identification, isolation and purification of proteinscontaining sulfotyrosine residues.

For example, the antibodies and antibody fragments of the invention willbe useful in a wide variety of immunological protein assays andisolation procedures, including without limitation, ELISAs, WesternBlots and other immunoblot techniques, immunohistochemical assays,various affinity purification methods, and the like. Accordingly, theinvention provides various immunological assays useful for the detectionof tyrosine sulfated proteins and for the detection of conditionscharacterized by the sulfation state of a particular protein orproteins. Such assays generally comprise one or more anti-sulfotyrosineantibodies capable of specifically recognizing and binding a tyrosinesulfated protein, and include various immunological assay formats wellknown in the art, including but not limited to various types ofprecipitation, agglutination, complement fixation, radioimmunoassays,enzyme-linked immunosorbent and immunofluorescent assays, enzyme-linkedassays, immunohistochemical analysis and the like.

The anti-sulfotyrosine antibodies of the invention may also be used totarget therapeutic targets. For example, an anti-sulfotyrosine antibodymay be conjugated to a toxin molecule and used to target the toxin to acell expressing a tyrosine sulfated protein.

The invention is further described by way of the following Exampleswhich illustrate the selection and characterization ofanti-sulfotyrosine antibodies using an scFv phage display system.Further details of the experimental work described therein may be foundin Kehoe, J. N., New Tools for Studying Tyrosine Sulfation, PhD Thesis,Univ. California, Berkeley 2004, the contents of which are incorporatedby reference herein.

EXAMPLES Example 1 Isolation of Sulfotyrosine-Specific Single ChainAntibodies

Libraries of phage-displayed single chain variable fragments (scFvs)containing both natural and unnatural combinations of heavy and lightchain variable regions were used to select scFvs recognizing tyrosinesulfate, using an in vivo recombination system for scFv production(Sblattero and Bradbury, 2000, Exploiting recombination in singlebacteria to make large phage antibody libraries. Nature Biotechnology18:75-80). Briefly, a small library of scFv was produced in a phagemidwith two non-homologous lox recombination. The small size of thislibrary makes it easy to construct and propagate. When one needs scFvsfor a selection, bacteria containing the phagemid library are infectedwith helper phage, and the resultant virions are used to infect bacteriawhich constitutively express the Cre recombinase. This infection is doneat a high multiplicity of infection, 200 virions per bacterium, toensure that multiple phage genomes enter each bacterium. The Crerecombinase is then able to shuffle the heavy and light chains of thescFvs in vivo, producing a much larger library of scFvs. After in vivorecombination, the recombinase-expressing bacteria are infected withhelper phage and the resultant virions collected. Since these bacteriacontained multiple phage genomes, one cannot be sure that the virion'sphenotype matches its genotype. A new set of bacteria must be infectedat a multiplicity of infection less then one. When these bacteria areinfected with helper phage the resultant virions will carry the gene forthe scFv they display, and can be used for selections.

Synthetic Peptide Antigens:

The antigens for scFv selection were based on two 17-residue peptideswith a tyrosine at the central position and a cysteine at the carboxyterminus (FIG. 1). Peptide one (Pep1) is loosely based on the tyrosinesulfation site of PSGL-1. As the goal was to develop an antibody thatbinds to every sulfated tyrosine rather than a sulfated tyrosine in aparticular sequence context, peptide 2 (Pep2) was also synthesized.

The two peptides were synthesized in sulfated and unsulfated forms (FIG.1). The non-sulfated peptides were synthesized using standardprocedures. HPLC analysis of the crude material showed that there wasone major product with few contaminants. The masses of the main productswere in close agreement with calculated values (Pep1: 2194.63calculated, 2195.20 observed; Pep2: 1907.26 calculated, 1906.8observed).

The tyrosine-sulfated peptides were synthesized manually, withN-hydroxybenzotriazole (HOBT) activated by2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU). After cleavage and deprotection of Pep1S, the peptide waspurified in the same manner as Pep1 and Pep2. The mass was as expected(2274.48 calculated, 2274.20 observed). HPLC purification of the cleavedand deprotected Pep2S showed a relatively clean reaction, but the mainproduct had the mass of a dimer. To reduce Pep2S to a monomer, the crudeproduct was dissolved in deionized water, a small amount oftris-carboxyethylphosphine (Pierce) was added, and the reaction held atroom temperature for seven hours. The peptide was then purified viareversed phase HPLC, and characterized by mass spectrometry (1989.26calculated, 1988.37 observed).

For use in a selection, a portion of the antigenic peptides werebiotinylated using EZ-link PEO iodoacetylbiotin (Pierce). The remainderwas coupled to the carrier proteins bovine serum albumin (BSA) orovalbumin (OVA) using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce). Theseconjugates were named using the peptide number, its sulfation state, andthe molecule to which it was bound. For example, Pep1S conjugated to BSAis BSA-1S. Pep2 conjugated to biotin will be referred to as Biotin-2.

Preliminary Selections:

Initial attempts to select a sulfotyrosine-specific scFv fromphage-displayed scFvs using antigens which had been adsorbed onto thesurface of an immunotube (IM) with a general elution methodology failed.Accordingly, the scFvs were first subjected to two preliminary tests:screening for sv5 expression and screening for binding to the backgroundcomponents of an IM selection. Screening for sv5 expression wasperformed to eliminate phage with genomic deletions, as phage are knownto frequently delete foreign DNA from their genome.⁵ The phage thatdelete the scFv gene are able to reproduce more rapidly than phage witha complete scFv gene, and selections can sometimes be overwhelmed byphage which do not carry a scFv gene.

The scFvs were analyzed for the presence or absence of the sv5 tagincorporated into the library construct using an immunoblot approach.Some 3810 colonies stemming from the initial selection attempt(non-stringent second round output and stringent third round output, inroughly equal proportions from selections against both peptide antigen,see Tables 1 and 2) were selected for this preliminary screen. Afterovernight growth to saturation, colonies were replicated ontoUV-sterilized nitrocellulose, and grown on a fresh plate of 2xYT agar,AmpGlu. After overnight growth, nitrocellulose was transferred to a 2xYTagar plate containing 0.25 mM IPTG to induce expression of the scFvs.The colonies were then lysed, and the nitrocellulose probed with mouseanti-sv5 and goat anti-mouse conjugated to alkaline phosphatase (Dako).Thirty percent of the second round output (non-stringent series) was sv5positive, while 39% of the third round output (stringent series) was sv5positive (Table 2). Overall, 34% of the clones were sv5 positive (Table2).

The sv5 positive scFvs were then tested for binding to a mixture of thecarrier proteins BSA and OVA. Using these two proteins as antigens, anELISA was run with crude scFv preparations, as a means of identifyingscFvs which bound to fish gelatin. A majority of the sv5 positive secondround output (non-stringent series) did not bind a background componentof the assay, while 66% of the third round output (stringent series) wasbackground negative.

Selections Against Sulfotyrosine Peptides:

Those sv5 positive scFvs which did not bind a background component ofthe ELISA (see above) were tested for binding to BSA-1, BSA-1S, BSA-2,and BSA-2S. Preliminary data indicated that 25 clones bound all four ofthese antigens. The linker between the peptide antigens and the carrierproteins was the only common structure between all four proteins, and itseems to have served as a very good antigen. Five scFvs bound Pep1regardless of sulfation state, and 2 bound Pep2 regardless of sulfationstate. Two clones appeared to bind Pep1S in a sulfate dependent manner.

Some 960 sv5 positive, background negative scFv clones appeared not tobind to anything used in the selections, and these were re-screenedusing an alkaline phosphatase reporter system in order to select furtheranti-sulfotyrosine scFvs. Alkaline phosphatase itself and the substrate,p-nitrophenylphosphate, are both quite stable under the conditions usedfor ELISAs. Therefore a weak ELISA signal will show linear growth for upto 15 hours with minimal background. The second batch of ELISAs was runin the same fashion, except that the tertiary antibody was a 1:2000dilution of goat α-mouse conjugated to alkaline phosphatase (Dako).

Seventy-four of these scFvs were background positive with alkalinephosphatase. The remaining scFvs were screened for binding to thesulfated antigens as described above. These screens uncovered anadditional seven scFvs which appeared to bind the peptide antigens in asulfate-dependent manner.

TABLE 1 Elution Strategy 1: stringent Selection Selection 1 Selection 23 Round 1 BSA-1S OVA-1S Biotin-1S Trypsin Round 2 OVA-2S Biotin-2SBSA-2S Trypsin Round 3 Biotin-1S BSA-1S OVA-1S Trypsin Strategy 2:non-stringent Selection Selection 4 Selection 5 6 Round 1 BSA-1S OVA-1SBiotin-1S Trypsin Round 2 OVA-1S Biotin-1S BSA-1S Trypsin Strategy 3:specific elution Selection Selection Selection 7 Selection 8 9 10 Round1 BSA-1S BSA-2S OVA-1S OVA-2S Ty sulfate Round 2 OVA-1S OVA-2S BSA-1SBSA-2S Ty sulfate Strategy 4: solution interaction Selection SelectionSelection Selection 11 12 13 14 Round 1 Biotin-1S Biotin-1S Biotin-2SBiotin-2S Trypsin Round 2 Biotin-1S Biotin-2S Biotin-1S Biotin-2STrypsin

TABLE 2 Sulfate Sulfate dependent dependent % binding - binding - No. %SV5 background phage scFv picked soluble negative ELISA ELISA Strategy1: 3810 1295 855 9 1 stringent Strategy 2: non-stringent Strategy 3: 573243 174 24 1 specific elution Strategy 4: 3528 2117 1863 0 solutioninteraction

Example 2 Characterization of Sulfotyrosine-Specific scFvs

Identification of Unique scFv Genes:

Genes encoding 33 scFvs specific for tyrosine sulfate (Example 1) wereamplified via PCR and digested with BstN I, a restriction site common inantibody variable domains. The resultant DNA fragments were separated ona polyacrylamide gel and visualized with ethidium bromide (see Table 2).These results indicated that 28 of the 33 clones produced a full-lengthscFv gene, while two appeared to be half the length of a normal scFvgene. All 30 of these clones gave unique restriction patterns upon BstNI digestion.

Expression of Unique scFvs:

The unique scFvs were expressed in E. coli and purified using standardprocedures (see, Kehoe, 2004, supra).

Binding Analysis Using Peptide Antigens:

Purified scFvs were then analyzed for specific binding to thesulfotyrosine peptide antigens. Briefly, immunosorb plates (Nunc) werecoated with either the sulfated or non-sulfated antigens and ELISAs wereconducted using scFv-conditioned media as a primary antibody source.Mouse anti-sv5 served as the secondary antibody and goat anti-mouseconjugated to alkaline phosphatase was the tertiary antibody. All scFvswere tested for their ability to bind BSA-1S and BSA-1, and most scFvswere tested for their ability to bind streptavidin-captured Biotin-2Sand Biotin-2.

The results are shown in FIG. 2. Two clones, clones scFv 25 and scFv 31,appear to bind specifically to the sulfated peptide antigens (FIG. 2C).

Binding Analysis Using Tyrosine-Sulfated Proteins:

The single chain antibody of clone scFv 25 was further analyzed forsulfotyrosine-specific binding using three commercially availabletyrosine-sulfated proteins: porcine thyroglobin, rat fibrinogen andmouse IgM (μ specific) (all SIGMA). Abalone sulfatase was used to removethe sulfate modification from the tyrosine residues in these proteins.This sulfatase was found not to degrade the proteins (FIG. 3). An ELISAprotocol was used to evaluate scFv 25 binding to each of these proteins,in both their sulfated and de-sulfated states.

The ELISA results are shown in FIG. 4 and indicate that scFv 25 bindsspecifically to the sulfated peptides and two of the three sulfatedproteins. A polyacrylamide gel electrophoresis of the proteins used inthe ELISA is shown in FIG. 5, and indicates the effects of sulfatasetreatment.

The nucleotide and translated amino acid sequences of scFv 25, and itscomponent V_(H) and V_(L) genes, and Lox linker sequences, are shown inthe TABLE OF SEQUENCES, infra.

Example 3 Generation of Anti-Sulfotyrosine IgG

This example describes the construction, expression and purification ofan anti-sulfotyrosine IgG using the VH and VK genes of clone scFv 25.Characterization of the resulting complete antibody is described inExample 4.

Materials and Methods:

IgG Generation: A full-length, complete IgG anti-sulfotyrosine antibodywas generated from scFv 25 as previously described (MacCallum et al.,1996, J. Mol. Biol. 262: 732-745). Briefly, the VH gene was amplifiedfrom its scfv expression phagemid clone with the primer pairs25STVH5′[GTA CCA ACG CGT GTC CAG TCT CAG GTG CAG CTG GTG GAG TCT] [SEQID NO: 13] and 25STVH3′[GTC TCC TGA GCT AGC TGA GGA GAC GGT GAC CAG GGT][SEQ ID NO: 14] by PCR, and the purified DNA fragment was digested withMluI and NheI, ligated into a human IgG1 expression vector N5KG1Val-Lark(a kind gift from Dr. Mitch Reff, IDEC Pharmaceuticals, San Diego) andclones containing the correct VH gene identified by DNA sequencing. TheVk gene of the clone was PCR amplified from the same phagemid vectorwith the primer pairs 25STVK5′ [TAC TCG CAG CM GCG GTG CAC GAT GTG CMTTG TGT TGA CAC AGT CTC C] [SEQ ID NO: 15] and 25STVK3′ [ATT ATA CGA AGTTAT GGT CGA CCC CGT ACG TTT GAT ATC CAC TTT GGT C] [SEQ ID NO: 16], andcloned into the pCR-2.1 vector (Invitrogen). Clones containing thecorrect Vk gene were identified by DNA sequencing. The Vk gene wasexcised from pCR-2.1 vector with DraIII and BsiWI and ligated intoDraIII and BsiWI-digested N5KG1Val-Lark DNA containing the appropriateVH gene. Clones containing the correct VH and Vk gene were identified byDNA sequencing, and vector DNA was used to transfect CHO DG44 cells byelectroporation. Stable cell lines were established by selection in G418and expanded into one liter spinner flasks. Supernatant containing IgGwas collected and purified on Protein G column (Pharmacia). The affinitypurified IgG was assessed by native and reduced SDS-PAGE, and proteinconcentration of the final stock was determined by A280 nm.

ELISAs: IgG ELISAs were conducted essentially as described in Example 2,supra, except that 1 μg/well IgG was used and the signal was detectedusing anti-human AP in 1:2000 dilution (Santa Cruz biotech).

Tyrosine Sulfate Competition ELISAs: Tyrosine sulfate competition ELISAswere conducted with both IgG and scFv. Bovine fibrinogen IV wasbiotinylated using the EZ link sulfo NHS LC-LC biotinylation kit(Pierce). 5□g of biotinylated antigen was incubated with 2 □g of scFv-APfor 1 hr in the presence or absence of competing compounds (tyrosinesulfate, tyrosine phosphate and tyrosine) at 5 mM. After incubation withantibody, the biotinylated antigen with bound scFv-AP was transferred tosuccessive wells using the Kingfisher magnetic particle processor(Thermoelectron). 10 □l of streptavidin coated magnetic beads (Dynal)were used for each sample and incubated for 10 min. 3×PBST and 3×PBSwashes were subsequently carried out and the AP signal after washingdetected using the phosphatase substrate kit (Pierce). The ELISA withthe IgG was carried out similarly, except that 5 μg of antigen and 1 □gof IgG were used, after the first wash, the complex was incubated with1:2000 dilution of anti-human AP for 1 hr and washed again prior tomeasurement. The absorbance at 405 nm reported represents the finalvalue obtained after background subtraction.

Western Blot Analyses: Antigens were separated by polyacrylamide gelelectrophoresis using a 4-12% gradient Novex acrylamide gel(Invitrogen), and electro-transferred onto nitrocellulose using asemi-wet electroblotter. The antigens were loaded in the followingamounts: —E. coli cell extract—30 □g, sulfatase—28 □g, the 3Fibrinogens—10 □g, C4—0.75 □g and vitronectin 10 □g. Prior to analysis,the blot was blocked using wonder block solution for at least 30minutes. 200 μg of IgG or 50 μg scFv-AP was diluted in 10 ml of 1×WB andincubated with the transferred blot for 1 hr. The blot was then washedfor 10 minutes with PBST (twice) followed by 10 minutes with PBS(twice). The bound IgG was dtetected using alkaline phosphatase labeledanti-human (Santa Cruz Biotech) antibodies after similar washings.Alkaline phosphatase activity was detected using NBT/BCIP (Pierce).

Results:

The IgG generated was subjected to charaterization using ELISA,competition ELISA and Western Blot.

ELISA with the IgG showed that it recognizes hirudin from leeches, threedifferent forms of fibrinogen (fraction 1s—bovine, fibrinogen fractionIV—bovine, fibrinogen-rat) and complement C4 (FIG. 6A), and that thesignal was lost upon sulfatase treatment. Interestingly, a correlationbetween the ELISA signals obtained for the different proteins and thenumber of sulfated tyrosines was noted: C4 and vitronectin gave thehighest ELISA signals and possessed three and two sulfated tyrosinesrespectively (see FIG. 1D). The scFv-AP conjugate was used to analyzebinding to human vitronectin (which could not be tested with the IgG asit gave a strong non-specific signal with the secondary anti-humanantibody) and confirm binding to human IgM (FIG. 6B). However, as can beseen from FIG. 6C, with the exception of IgM and C4, there is againsignificant proteolysis upon sulfatase treatment.

In order to further demonstrate the specificity of the antibody for thetyrosine sulfate modification, a magnetic bead based ELISA (KingFisher,Thermoelectron Inc.) was developed with the goal to see if antibodybinding could be inhibited by soluble tyrosine sulfate. Biotinylatedbovine fibrinogen IV, the anti-sulfotyrosine antibody and PBS, 5 mMtyrosine sulfate, 5 mM tyrosine phosphate or 5 mM tyrosine wereincubated together for an hour. After this period, the amount ofantibody which was bound to the biotinylated fibrinogen was assessed byadding streptavidin magnetic beads followed by washing. As can be seenin FIG. 7A, incubation with 5 mM tyrosine sulfate reduced the signal tobackground levels, while tyrosine phosphate and tyrosine had no effectwhatsoever, indicating the specificity of the binding. Similar resultswere obtained with the scFv-AP fusion (FIG. 7B, and see Example 2), anda control experiment with lysozyme and an anti-lysozyme antibody showedthat signal inhibition was not due to non-specific inhibitory effectscaused by tyrosine sulfate. This inhibition was further studied bytitrating the amount of tyrosine sulfate required to inhibit binding. Ascan be seen in FIG. 7C, at 10 mM tyrosine sulfate, almost fullinhibition is observed, while half maximal inhibition is seen atapproximately 1.25 mM.

The question of whether the antibody was able to recognize the tyrosinesulfate modification was also assesed by Western Blot. Analyses with thevarious fibrinogens and C4 were carried out with the IgG followed byanti-human AP, while the vitronectin was assayed using the scFv-APconjugate, as it gave a very high background with the secondaryantibody. Each of the proteins is clearly recognized by theanti-tyrosine sulfate antibody, with a signal intensity not correlatedto the intensity of the coomassie blue-band, suggesting that the visiblebands are differentially sulfated. These proteins were treated with theabalone sulfatase, modulated to reduce proteolysis for each protein(FIG. 8A). Following sulfatase treatment, the Western blot signal foreach protein is abolished or significantly reduced (FIG. 8B), withoutaffecting the integrity of the protein itself. As E. coli does notexpress any sulfated proteins, lacking the enzymatic machinary to do so(Moore, K. L., 2003, J. Biol. Chem. 278: 24243-24246), the extent ofnon-specific binding of the antibody was evaluated by probing anoverloaded E. coli extract. As can be seen in FIG. 8B, lane 1, no signalis obtained whatsoever, indicating that the antibody is unable to reactwith the diverse array of E. coli proteins expressed under normal growthconditions.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE OF SEQUENCES: scFv 25 NUCLEOTIDE SEQUENCE [VL-lox linker-VH] SEQID NO: 1 GCAATTGTGTTGACACAGTCTCCATCGTCCCTGTCTGCCTCTGTCGGAGACAGAGTCATCATCACTTGCCGGGCAAGTCAGAGTATTACTAAATATGTAAATTGGTATCAGCAAAAACCAGGAAAGGCCCCTAACCTCCTCATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTGCAACCTGAGGACTTTGCAACCTACTACTGTCAGCAGACTTACAATGTCCCTCGGACGTTCGGCCAAGGGACCAAAGTGGATATCAAATCCGGAGGGTCGACCATAACTTCGTATAATGTATACTATACGAAGTTATCCTCGAGCGGTACCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATCCACGTATGATAGTAGTGGTTATTACCGGCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCAC CGTCTCCTCA scFv 25TRANSLATED AMINO ACID SEQUENCE [VL-lox linker-VH] SEQ ID NO: 2IVLTQSPSSLSASVGDRVIITCRASQSITKYVNWYQQKPGKAPNLLIYGASSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQTYNVPRTFGQGTKVDIKSGGSTITSYNVYYTKLSSSGTQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTYDSSGYYRHWYFDLWGRGTLVTV SS NUCLEOTIDESEQUENCE OF VARIABLE LIGHT CHAIN GENE OF scFv 25 SEQ ID NO: 3GCAATTGTGTTGACACAGTCTCCATCGTCCCTGTCTGCCTCTGTCGGAGACAGAGTCATCATCACTTGCCGGGCAAGTCAGAGTATTACTAAATATGTAAATTGGTATCAGCAAAAACCAGGAAAGGCCCCTAACCTCCTCATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTGCAACCTGAGGACTTTGCAACCTACTACTGTCAGCAGACTTACAATGTCCCTCGGACGTTCGGCCAA GGGACCAAAGTGGATATCAAAAMINO ACID SEQUENCE OF VARIABLE LIGHT CHAIN COMPONENT OF scFv 25 SEQ IDNO: 4 IVLTQSPSSLSASVGDRVIITCRASQSITKYVNWYQQKPGKAPNLLIYGASSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQTYNVPRTFGQG TKVDIK NUCLEOTIDESEQUENCE OF LOX LINKER COMPONENT OF scFv 25 SEQ ID NO: 5TCCGGAGGGTCGACCATAACTTCGTATAATGTATACTATACGAAGTTATC CTCGAGCGGTACC AMINOACID SEQUENCE OF LOX LINKER COMPONENT OF scFv 25 SEQ ID NO: 6SGGSTITSYNVYYTKLSSSGT NUCLEOTIDE SEQUENCE OF VARIABLE HEAVY CHAIN GENEOF scFv 25 SEQ ID NO: 7CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATCCACGTATGATAGTAGTGGTTATTACCGGCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCA AMINO ACID SEQUENCE OF VARIABLE HEAVY CHAINCOMPONENT OF scFv 25 SEQ ID NO: 8QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTYDSSGYYRHWYFDLWGRGTLVTVSS AMINO ACID SEQUENCE OF SYNTHESTIC PEPTIDE Pep1SEQ ID NO: 9 KDKKYATEYEYLDYDFC AMINO ACID SEQUENCE OF SYNTHESTIC PEPTIDEPep1S [asterisk indicates SO₃ ⁻  modification of Tyrosine residue] SEQID NO: 10 KDKKYATEY^(*)EYLDYDFC AMINO ACID SEQUENCE OF SYNTHESTICPEPTIDE Pep2 SEQ ID NO: 11 KAKISDPDYMTGYMDAC AMINO ACID SEQUENCE OFSYNTHESTIC PEPTIDE Pep2S [asterisk indicates SO₃ ⁻  modification ofTyrosine residue] SEQ ID NO: 12 KAKISDPDY^(*)MTGYMDAC

1-24. (canceled)
 25. An isolated polynucleotide encoding a sulfotyrosineantibody, which antibody is specific for a sulfated tyrosine antigenicdeterminant in a sulfotyrosine-containing polypeptide and comprises aheavy chain variable region having the amino acid sequence of SEQ ID NO:8 and a light chain variable region having the amino acid sequence ofSEQ ID NO:
 4. 26. The isolated polynucleotide of claim 25, which encodesa full length immunoglobulin selected from the classes consisting ofIgA, IgD, IgE, IgG and IgM.
 27. An expression vector comprising apolynucleotide according to claim
 25. 28. An expression vectorcomprising a polynucleotide according to claim
 26. 29. A host cellcomprising the vector of claim
 27. 30. A host cell comprising the vectorof claim 28.